Nitrogen trifluoride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Short description of key information:

In accordance with column 1 section 8.7.3 of REACH annex IX, a two-generation reproductive toxicity study is not deemed necessary as no adverse effects on the reproductive organs were found in the inhalation 90-day study (OECD 413) with NF3, where complete histopathology of the sex organs was undertaken

Justification for selection of Effect on fertility via oral route:

As nitrogen trifluoride is a gas, the oral route of exposure is not relevant

Justification for selection of Effect on fertility via inhalation route:

In accordance with column 1 section 8.7.3 of REACH annex IX, a two-generation reproductive toxicity study is not deemed necessary as no adverse effects on the reproductive organs were found in an inhalation 90-day study (OECD 413) with NF3, where complete histopathology of the sex organs was undertaken.  Therefore, the NOAEC for reproductive toxicity has been taken from the 90-day inhalation study, where the highest dose tested (100 ppm) showed no adverse effects on the reproductive organs.

Justification for selection of Effect on fertility via dermal route:

As nitrogen trifluoride is a gas, the dermal route of exposure is not relevant

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Quality of whole database:

n/a

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEC

296 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

90-d repeat dose inhalaltion study: - KL.1

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

n/a

Additional information

No suitable studies on NF₃ have been identified to address the endpoint of reproductive toxicity (fertility and sexual function), however in accordance with column 1, Section 8.7.3 of the REACh annex IX guidance a two generation reproductive toxicity study is not deemed necessary as no adverse effects on the reproductive organs were found in the 90 -day inhalation study (OECD 413 - Section 7.5.2: KS: O'Neill (2003). KL.1) with NF3, where complete histopathology of the sex organs were undertaken.

Effects on developmental toxicity

Description of key information

Justification for selection of Effect on developmental toxicity: via inhalation route:

The study by Tyl et al (1987) provides a weight of evidence approach for MetHb forming chemicals and their lack of developmental effects

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, but comparable with the requirements of the TG with exposure limited to the period of organogenesis Read-across approach based on similarities in the mode of toxicological action - through the formation of methaemoglobin

Justification for type of information:

REPORTING FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As few data are available on the target substance nitrogen trifluoride (CAS 7783-54-2 / EINECS 232-007-1). A research of the potential analogues has been carried out.
In order to fulfil the read-across requirement detailed above, the hypothesis is that properties are likely to be similar or follow a similar pattern as a result of the presence of a common structure. This is a reasonable assumption for the majority of inorganic compounds and some organic compounds (e.g. fluorocarbons compounds).
The following points are considered:
- Chemical speciation and structure (nitrogens compounds) which are simple and similar.
- In fluids of organisms and in aqueous media, benzene cycle easily dissociates from nitrogen function. Toxicity of nitrobenzene is thus considered as covering toxicity due to both benzene and nitrogen function.
- In the case where we should consider dissociation or breakdown products, the common structure of nitrogen function should lead to common breakdown products. Thus, the eventual toxicology related to this function should be considered equally and with similar mode of action.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source is nitrogen trifluoride (CAS 7783-54-2 / EINECS 232-007-1).
The analogue identified is nitrobenzene.
According to the regulation data available in the ECHA website and the concentration of impurities, impurities have no impact on the classification of the nitrogen trifluoride produced by the LEAD REGISTRANT.

3. ANALOGUE APPROACH JUSTIFICATION
Analogues were identified according to the criteria exposed in the section 1 (hypothesis for analogue approach) presented above. Then, data on these analogues were analyzed and they are presented in the datamatrix in the document attached. Physico-chemical and global toxicological properties (taking into consideration classification and available toxicological information) are considered similar to the target chemical and thus validate the analogue approach.

4. DATA MATRIX
See document attached.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

exposure limited to the period of organogenesis

GLP compliance:

no

Limit test:

no

Strain:

Sprague-Dawley

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs of toxicity observed were thoses typical of inhalation studies: occasional localized alopecia, slight erosion of the tail tip, and, rarely, periocular wetness; there were no exposure- related or concentration-related signs of toxicity

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

unaffected by treatment. However, maternal weight gain (a more sensitive measure) was significantly depressed relative to controls at 40 ppm, for days 6-9 (-61%) and 6-15 (-19%) of the treatment period. Weight gains for the latter part of the exposure period (days 9-12 and 12-15) were unaffected by treatment. Weight gain during the post-exposure period (days 15-18 and 15- 21) was significantly elevated at 40 ppm relative to controls by 20% and 13%, respectively, so that maternal body weight at sacrifice (day 21) was equivalent across all groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects of exposure on body weight (absolute or corrected for gravid uterine weight), on gravid uterine weight, on kidney weights (absolute or relative). Absolute and relative liver weights were increased at 40.0 ppm (103.6 and 104.6% of controls, respectively) but the differences were not statistically significant. Spleen weights (absolute and relative) were significantly elevated at 10.0 ppm (+15% and +14%) and at 40.0 ppm (+40% and +41%) with a clear exposure level response.

Number of abortions:

no effects observed

Description (incidence and severity):

Gestational parameters were unaffected by treatment. The control and treatment groups did not differ in number ofcorpora lutea/dam; in total, non-viable (resorptions and dead fetuses) and viable implantations (live fetuses)/litter; in percentage pre- or postimplantation loss; in sex ratio (% males); or in fetal body weight (males, females, all fetuses)/litter

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Dose descriptor:

NOAEC

Effect level:

ca. 1 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no significant increase in the number of litter with one or more affected fetuses at any exposure concentration relative to controls for individual and total external, visceral (including craniofacial), or skeletal malformations

External malformations:

effects observed, treatment-related

Description (incidence and severity):

There was a significant increase of +33% in the incidence of total malformations (but not in the incidence of any individual malformations or of malformations by category) at 10 ppm but not at 10.0 or 40.0 ppm relative to that of controls.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There was no significant increase in the number of litter with one or more affected fetuses at any exposure concentration relative to controls for individual and total external, visceral (including craniofacial), or skeletal malformations

Visceral malformations:

no effects observed

Description (incidence and severity):

There was no significant increase in the number of litter with one or more affected fetuses at any exposure concentration relative to controls for individual and total external, visceral (including craniofacial), or skeletal malformations

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEC

Effect level:

> 40 ppm

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

FETAL EFFECTS:

There were 9 external, 52 visceral (including craniofacial), and 139 skeletal findings that were classified as variations. Examples of external variations observed included ecchymosis of head and extremities and bruises on trunk; examples of visceral variations were shortened innominate artery, irregular rugae on palate, fetal atelectasis, red foci on thymus, and dilated ureters; examples of skeletal variations included poorly ossified cervical and thoracic centra, sternebrae 2, 5, and 6, and skull plates. The incidence of five of these variations (one external, four skeletal) was significantly different for one of the nitrobenzene-exposed groups from that of controls. The incidence of litters with one or more fetuses with external variations was significantly elevated by 64% at 40.0 ppm for ecchymoses on the trunk (but not on the head or extremities). There were no effects of treatment on the incidence of visceral variations. The incidence of four skeletal variations in nitrobenzene-exposed groups differed significantly from that of controls for the following: The incidence of split (bipartite) anterior arch of the atlas was significantly elevated by +600% at 1.0 ppm but not at 10.0 or 40.0 ppm), the incidence of bilobed thoracic centrum 9 was significantly reduced at 40.0 ppm by -83%, the incidence of parietal skull plate with a "hole" in bone (defined as a precisely delineated area of nonossification surrounded by ossification) was elevated at 40.0 ppm by +138%, and the incidence of poorly ossified premaxillary bone was significantly elevated at 1.0 ppm by +267%.

 

Table 7.8.2-01: Maternal body weight gain:

	0 ppm	1 ppm	10 ppm	40 ppm
% pregnant females at sacrifice	96.2	96.2	96.2	100
DAYS	Body weight gain (g)
Pre-exposure period (GD 0-6)	28.5 ± 6.6	27.9 ± 7.9	29.6 ± 6.6	27.2 ± 5.8
GD 6-9	11.8 ± 8.4	10.0 ± 3.9	9.5 ± 6.0	4.6 ± 7.2***
GD 9-12	11.7 ± 9.0	14.0 ± 5.2	13.2 ± 4.8	12.2 ± 7.4
GD12-15	15.0 ± 6.4	13.5 ± 3.9	16.9 ± 4.0	14.7 ± 6.8
Post exposure period (GD 6-15)	38.6 ± 7.0	36.8 ± 6.9	39.6 ± 6.3	31.4 ± 7.6**
Post exposure period (GD 15-18)	29.3 ± 935	32.5 ± 6.7	33.1 ± 4.6	35.2 ± 5.9**
Post exposure period (GD 18-21)	44.8 ± 10.2	47.1 ± 8.6	48.5 ± 7.0	48.5 ± 6.6
Overall entire post exposure period (GD15-21)	74.2 ± 13.7	79.7 ± 12.1	81.6 ± 8.9	83.8 ± 8.4**

**p<0.01; ***p<0.001

 

Table 7.8.2-02: Maternal organ weights at sacrifice:

	0 ppm	1 ppm	10 ppm	40 ppm
Body weight at sacrifice (g)	371.8 ± 23.2	374.9 ± 22.6	381.3 ± 21.1	373.3 18.6
Corrected body weight (= body weight at sacrifice - gravid uterine weight; g)	273.53 ± 16.35	274.25 ± 15.51	280.42 ± 14.29	271.07 13.03
Gravid uterine weight (g)	98.25 ± 16.34	100.65 ± 12.73	100.86 ± 12.51	102.24 10.80
Liver weight (g)	13.55 ± 1.31	13.35 ± 1.79	13.78 ± 1.66	14.04 1.24
Relative liver weight (%)	4.95 ± 0.36	4.86 ± 0.49	4.90 ± 0.45	5.18 0.35
Kidney weight (g)	1.91 ± 0.17	1.84 ± 0.14	1.88 ± 0.17	1.87 0.10
Relative kidney weight (%)	0.70 ± 0.06	0.67 ± 0.04	0.67 ± 0.06	0.69 0.04
Spleen weight (g)	0.60 ± 0.10	0.60 ± 0.11	0.69 ± 0.12*	0.84 0.12***
Relative spleen weight (%)	0.22 ± 0.03	0.22 ± 0.04	0.25 0.04*	0.31 0.04***

*p<0.05;***p<0.001

 

Table 7.8.2-03: Malformations observed in fetuses exposedin utero

	0 ppm	1 ppm	10 ppm	40 ppm
External malformations	1 (1)	1 (1)	0 (0)	0 (0)
Visceral malformations	8 (5)	14 (7)	3 (3)	17 (7)
Skeletal malformations	41 (16)	58(21)	46 (15)	46 (15)
Total malformations	49 (18)	72 (24*)	49 (17)	63 (20)

*p<0.05

Values in parenthesis refer to litter data

 

Table 7.8.2-04: Significant variations in fetuses

	0 ppm	1 ppm	10 ppm	40 ppm
Number examined externally	347 (25)	353 (25)	361 (25)	377 (26)
Ecchymosis on trunk	21 (14)	27 (17)	34 (17)	38 (23*)
Number examined viscerally	180 (25)	181 (25)	187 (25)	196 (26)
Number examined skeletally	167 (25)	172 (25)	174 (25	181 (26)
Anterior arch of atlas, split	1 (1)	7 (7*)	5 (5)	6 (5)
Thracic centrum 9, bilobed	6 (6)	3 (3)	3 (3)	1 (1*)
Parietal, hole in bone	9 (8)	15 (9)	21 (11)	29 (19)*
Premaxiliary, poorly ossified	3 (3)	19 (11)	13 (7)	12 (6)

*p<0.05

Values in parenthesis refer to litter data

Executive summary:

In the absence of relevant experimental data to determine the developmental toxicological properties of NF3, a read-across approach has been taken using nitrobenzene which has a similar mode of toxicological action as NF3, i.e. through the formation of methaemoglobin. The read-across approach adopted is according to Annex XI of regulation 1907/2006/EC (REACh regulation). Existing data for nitrobenzene was evaluated by the Registrant and deemed to fulfil the developmental toxicity endpoint requirement. The information provided here represents a scientifically sound justification. The Registrant considers the given arguments as being appropriate for evaluating the developmental toxicity endpoint and waiving further studies with vertebrate animals. This is in accordance with Article 25 of the REACh regulation, which states that testing on vertebrate animals shall be considered only as a last resort.

 

Pregnant CD (Sprague-Dawley) rats were exposed to nitrobenzene vapor (CAS Registry No. 98-95-3) at 0, 1, 10, and 40 ppm (mean analytical values of 0.0, 1.06, 9.8, and 39.4 ppm, respectively) on gestational days (gd) 6 through 15 for 6 hr/day. At sacrifice on gd 21, fetuses were evaluated for external, visceral, and skeletal malformations and variations. Maternal toxicity was observed: weight gain was reduced during exposure (gd 6-9 and 6-15) to 40 ppm, with full recovery by gd 21, and absolute and relative spleen weights were increased at 10 and 40 ppm. There was no effect of treatment on maternal liver, kidney, or gravid uterine weights, on pre- or post-implantation loss including resorptions or dead fetuses, on sex ratio of live fetuses, or on fetal body weights (male, female, or total) per litter. There were also no treatment-related effects on the incidence of fetal malformations or variations. In summary, during organogenesis in CD rats, there was no developmental toxicity (including teratogenicity) associated with exposure to nitrobenzene concentrations that produced some maternal toxicity (10 and 40 ppm) or that produced no observable maternal toxicity (1 ppm).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

Whilst the study is not GLP compliant, the conduct of the study was comparable to the requirements of the TG.

Additional information

In the absence of relevant experimental data to determine the developmental toxicological properties of NF₃, a read-across approach has been taken using nitrobenzene which has a similar mode of toxicological action as NF₃, i.e. through the formation of methaemoglobin. The read-across approach adopted is according to Annex XI of regulation 1907/2006/EC (REACh regulation). Existing data for nitrobenzene was evaluated by the Registrant and deemed to fulfil the developmental toxicity endpoint requirement. The information provided here represents a scientifically sound justification. The Registrant considers the given arguments as being appropriate for evaluating the developmental toxicity endpoint and waiving further studies with vertebrate animals. This is in accordance with Article 25 of the REACh regulation, which states that testing on vertebrate animals shall be considered only as a last resort.

 

NF3 is known to form MetHb anaemia, resulting in Hb becoming oxidised and rendering its ability to carry oxygen and consequently destroy RBC. Therefore, in the absence of such experimental data a read-across approach is proposed. Ideally this read-across approach would be drawn from chemicals which possess similar chemical / physical properties to that of NF3; however the required quality of data to address this endpoint is not available in the public domain, so the read-across approach has been constructed through the mode of toxicological action - the formation of MetHb. 

 

Nitrobenzene, like NF3 also exerts its toxic mode of action through the formation of MetHb. A complete developmental toxicity study (including soft tissue and skeletal examinations) in rats has been undertaken with nitrobenzene, which was administered via the inhalatory route. No evidence of developmental effects following exposure to nitrobenzene were observed, this included doses which showed evidence of maternal toxicity.

 

Data in the literature would suggest that inhibition of oxygen binding through the formation of MetHb does not show evidence of developmental toxicity. This is stark contrast to CO which whilst also known to inhibit oxygen binding through the formation of carboxyhaemoglobin (COHb) formation which is known to be both fetotoxic and teratogenic. The mechanism of formation of COHb, like MetHB is reversible. Studies with CO where skeletal examinations have taken place show significant increases in both gross and skeletal malformations and limb deformities (Singh et al, 1992). CO is also known to target the heartin utero. These two studies in particular found maternal exposure to CO to be associated with an increased risk of cardiac birth defects, which is mirrored in human clinical studies also demonstrating the heart as a target for CO effects (EHC, 1999 and EPA, 2010). In conclusion the data would imply that inhibition of oxygen binding to Hb, through the formation of MetHb does not cause developmental effects, unlike COHb formation.

 

As NADH-methaemoglobin reductase is deficient in neonates they are especially sensitive to chemicals that cause methaemoglobinaemia (Gregus & Klassen, 2001). NADH lacks full activity until infants are 4 months of age. Whilst this is important to note, this has no impact on NF3 for the uses proposed.

Justification for classification or non-classification

NF3 is considered to be neither a reproductive toxicant (based on the 90 -day inhalation histopathology data generated on the reproductive organs) nor a developmental toxicant (based on a read-across approach to nitrobenzene). Therefore classification is not required.